Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
The kidney develops from two components, the metanephric mesenchyme and the ureteric bud. The ureteric bud invades the mesenchyme, triggering both mesenchymal proliferation and the conversion of the mesenchyme into epithelia. This process has been termed induction of the mesenchyme (Grobstein, 1955; Saxen, 1987; Ekblom, 1989). The bud is known to produce factors which stimulate growth of the metanephric mesenchyme (Perantoni et al., 1995; Barasch et al., 1997), but the ureteric factors which convert mesenchyme into epithelia have been unknown. To date, induction of isolated metanephric mesenchyme in vitro has been obtained with living cells, including a number of embryonic tissues (Grobstein, 1955; Sariola et al., 1989; Herzlinger et al., 1994; Kispert et al., 1998), but not with purified molecules.
To identify ureteric factors that trigger mesenchymal induction, we developed ureteric bud cell lines (UB cells; Barasch et al., 1996). These cells express a number of proteins in common with the embryonic ureteric bud at the time it invades the mesenchyme, including epithelial specific proteins (E-cadherin, ZO-1, cytokeratin, collagen IV, and laminin), receptor tyrosine kinases (c-ret and c-met), lectin (dolichos bifloris) binding sites (Barasch et al., 1996; Sakuri et al., 1997), as well as several monoclonal binding sites which cross react with the ureteric bud. In addition, UB cells secrete a number of metanephric mesenchymal growth factors (FGF-2, FGF-9, TIMP-1 and TIMP-2) that are also synthesized by the embryonic ureteric bud (Barasch et al, 1997; Barasch et al., 1999). We previously found that UB cells secrete factors (Barasch et al., 1997) that stimulate mesenchymal growth but cannot trigger metanephric mesenchymal conversion to epithelia.
The present application discloses the following. UB cells secrete several activities that in the presence of mesenchymal growth factors (Karavanova et al., 1996) trigger mesenchymal to epithelial conversion and tubulogenesis.
Purification and sequencing of one activity identified leukemia inhibitory factor (LIF). Metanephric mesenchymes treated with recombinant LIF (rLIF) were converted into epithelia and developed tubules as well as early nephrons. Other cytokines that signal through the gp130 receptor, like LIF, were also shown to induce epithelia. In vivo, LIF is expressed by the ureteric bud during invasion of the metanephric mesenchyme, while LIF receptors are expressed by the mesenchyme. Thus, as disclosed in the present application, LIF is a candidate mediator of epithelialization and morphogenesis in the embryonic kidney.